Cotransfection and Expression of LIM mineralization protein-1 and -3 in Mesenchymal Stem Cells in vitro_West China Medical Journal

Authors：

TANG Chunhui , TANG Xudong , LAI Tieying

Department of Orthopedics, the First Hospital of Neijiang City, Neijiang, Sichuan 641000, P. R. China;

Corresponding?author：

Keywords：

LIM礦化蛋白-1; LIM礦化蛋白-3; 質粒pEGFP-N2; 骨髓間充質干細胞; 兔

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【摘要】　目的　探討LIM礦化蛋白（LIM mineralization protein，LMP）-1和LMP-3雙基因共轉染骨髓間充質干細胞（bone mesenchymal stem cells，BMSC）的表達情況。　方法　采用人工設計合成人LMP-1和LMP-3基因片段，分別與質粒pEGFP-N2連接，經酶切、測序鑒定后。分離培養新西蘭兔BMSC，用脂質體包裹轉染BMSC，按轉染情況分為5組:未轉染組（A組）、轉染空載體組（B組）、轉染LMP-1基因組（C組）、轉染LMP-3基因組（D組）、LMP-1與LMP-3雙基因共轉染組（E組）。采用實時聚合酶鏈反應（real-time polymerase chain reaction，RT-PCR）和蛋白質印跡法檢測LMP-1和LMP-3的表達。　結果　酶切及測序表明真核表達質粒pEGFP-N2-LMP-1和pEGFP-N2-LMP-3構建成功。E組可同時較高水平表達LMP-1和LMP-3分子。對RT-PCR及蛋白質印跡法檢測結果行灰度值測量并行統計學分析顯示:LMP-1 mRNA及蛋白水平的表達，5組間差異有統計學意義（P lt;0.05），但E組與C組的差異無統計學意義（P gt;0.05）；LMP-3 mRNA及蛋白水平的表達，5組間差異有統計學意義（P lt;0.05），且E組與D組差異也有統計學意義（P lt;0.05）。　結論　雙基因共轉染的BMSC能在體外同時表達LMP-1與LMP-3，為基因修復骨缺損帶來新思路。
【Abstract】　Objective　 To study the expression of LIM mineralization protein (LMP)-1 and LMP-3 genes after cotransfecting them into bone mesenchymal stem cells (BMSC) of rabbit in vitro. 　Methods　 Fragments of LMP-1 gene and LMP-3 gene were gained through artificial synthesis, and were constructed respectively into the plasmid vector pEGFP-N2. The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes. The plasmids carrying LMP-1 and LMP-3 genes were cotransfected into chondrocytes by liposome method. According to the transfected situation, the BMSC were divided into 5 groups: the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by LMP-1 (Group C), the group transfected by LMP-3 (Group D) and the group transfected by both LMP-1 and LMP-3 (Group E). The expressions of LMP-1 and LMP-3 were detected by RT-PCR and western bloting technique.　Results　 The plasmid pEGFP-N2-LMP-1 and pEGFP-N2-LMP-1 were obtained successfully by cloning technique and verified by nucleotide sequencing and enzymes. The LMP-1 and LMP-3 molecules were both expressed at a high level in Group E. The results of RT-PCR and western bloting were measured with the grey value. For the expression of LMP-1 mRNA and protein of LMP-1, the differences between groups A, B and groups C, D, E were significant (P＜0.05), while the difference between groups C and E was not significant (P＞0.05); For the expression of LMP-3 mRNA and protein of LMP-3, the differences between groups A, B and groups C, D, E were significant (P＜0.05), and the difference between groups D and E was also significant（P＜0.05).　Conclusion　 LMP-1 and LMP-3 genes can be expressed effectively after being cotransfected into BMSC, which provides a basis for gene therapy for treating bone defects.

Citation： TANG Chunhui,TANG Xudong,LAI Tieying. Cotransfection and Expression of LIM mineralization protein-1 and -3 in Mesenchymal Stem Cells in vitro. West China Medical Journal, 2011, 26(9): 1331-1335. doi: Copy

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2.	Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells[J]. J Cell Physiol, 1998, 176(1): 57-66.
3.	Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science, 1999, 284(5411): 143-147.
4.	Kassem M, Abdallah BM. Human bone-marrow-derived mesenchymal stem cells: biological characteristics and potential role in therapy of degenerative diseases[J]. Cell Tissue Res, 2008, 331(1): 157-163.
5.	Minamide A, Boden SD, Viggeswarapu M, et al. Mechanism of bone formation with gene transfer of the cDNA encoding for the intracellular protein LMP-1[J]. J Bone Joint Surg Am, 2003, 85-A(6): 1030-1039.
6.	Pola E, Gao W, Zhou Y, et al. Efficient bone formation by gene transfer formation by gene transfer of human LIM mineralization protein-3[J]. Gene Ther, 2004, 11(8): 683-693.
7.	Zhang Q, Wang X, Chen Z, et al. Semi-quantitative RT-PCR analysis of LIM mineralization protein-1 and its associated molecules in cultured human dental pulp cells[J]. Arch Oral Biol, 2007, 52(8): 720-726.
8.	Boden SD, Titus L, Hair G, et al. Lumbar spine fusion by local gene therapy with a cDNA encoding a novel osteoinductive protein (LMP-1)[J]. Spine (Phila Pa 1976), 1998, 23(23): 2486-2492.
9.	程志安, 劉冬斌, 吳燕峰, 等, 重組腺病毒載體Ad-LMP-1的構建及其轉染MSC后成骨活性[J]. 中山大學學報(醫學科學版), 2010, 31(2): 199-206.
10.	楊渝勇, 倪衛東. LMP-3功能區真核表達質粒的構建[J]. 重慶醫科大學學報, 2008, 5(33): 596-599.
11.	Kim HS, Viggeswarapu M, Boden SD, et al. Overcoming the immune response to permit ex vivo gene therapy for spine fusion with human type 5 adenoviral delivery of the LIM mineralization protein-1 cDNA[J]. Spine (Phila Pa 1976), 2003, 28(3): 219-226.
12.	李修洋, 鄧忠良, 徐希彥, 等. 人LMP-1基因腺病毒重組體構建及其在骨髓間充質干細胞的表達[J]. 中國組織工程研究與臨床康復, 2008, 12(21): 4084-4088.

1. 　Boden SD, Liu Y, Hair GA, et al. LMP-1, a LIM-domain protein, mediates BMP-6 effects on bone formation[J]. Endocrinology, 1998, 139(12): 5125-5234.
2. 　Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells[J]. J Cell Physiol, 1998, 176(1): 57-66.
3. 　Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science, 1999, 284(5411): 143-147.
4. 　Kassem M, Abdallah BM. Human bone-marrow-derived mesenchymal stem cells: biological characteristics and potential role in therapy of degenerative diseases[J]. Cell Tissue Res, 2008, 331(1): 157-163.
5. 　Minamide A, Boden SD, Viggeswarapu M, et al. Mechanism of bone formation with gene transfer of the cDNA encoding for the intracellular protein LMP-1[J]. J Bone Joint Surg Am, 2003, 85-A(6): 1030-1039.
6. 　Pola E, Gao W, Zhou Y, et al. Efficient bone formation by gene transfer formation by gene transfer of human LIM mineralization protein-3[J]. Gene Ther, 2004, 11(8): 683-693.
7. 　Zhang Q, Wang X, Chen Z, et al. Semi-quantitative RT-PCR analysis of LIM mineralization protein-1 and its associated molecules in cultured human dental pulp cells[J]. Arch Oral Biol, 2007, 52(8): 720-726.
8. 　Boden SD, Titus L, Hair G, et al. Lumbar spine fusion by local gene therapy with a cDNA encoding a novel osteoinductive protein (LMP-1)[J]. Spine (Phila Pa 1976), 1998, 23(23): 2486-2492.
9. 　程志安, 劉冬斌, 吳燕峰, 等, 重組腺病毒載體Ad-LMP-1的構建及其轉染MSC后成骨活性[J]. 中山大學學報(醫學科學版), 2010, 31(2): 199-206.
10. 　楊渝勇, 倪衛東. LMP-3功能區真核表達質粒的構建[J]. 重慶醫科大學學報, 2008, 5(33): 596-599.
11. 　Kim HS, Viggeswarapu M, Boden SD, et al. Overcoming the immune response to permit ex vivo gene therapy for spine fusion with human type 5 adenoviral delivery of the LIM mineralization protein-1 cDNA[J]. Spine (Phila Pa 1976), 2003, 28(3): 219-226.
12. 　李修洋, 鄧忠良, 徐希彥, 等. 人LMP-1基因腺病毒重組體構建及其在骨髓間充質干細胞的表達[J]. 中國組織工程研究與臨床康復, 2008, 12(21): 4084-4088.

West China Medical Journal

Cotransfection and Expression of LIM mineralization protein-1 and -3 in Mesenchymal Stem Cells in vitro

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